Multilayered cell culture apparatus

ABSTRACT

A multilayered cell culture apparatus for the culturing of cells is disclosed. The cell culture apparatus is defined as an integral structure having a plurality of cell culture chambers in combination with tracheal space(s). The body of the apparatus has imparted therein gas permeable membranes in combination with tracheal spaces that will allow the free flow of gases between the cell culture chambers and the external environment. The flask body also includes an aperture that will allow access to the cell growth chambers by means of a needle or cannula. The size of the apparatus, and location of an optional neck and cap section, allows for its manipulation by standard automated assay equipment, further making the apparatus ideal for high throughput applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/812,671, filed on Jul. 29, 2015, which is a continuation of U.S.application Ser. No. 14/699,157, filed on Apr. 29, 2015, which is acontinuation of U.S. application Ser. No. 14/075,446, filed on Nov. 8,2013, which is a continuation of U.S. application Ser. No. 13/904,171,filed on May 29, 2013, which is a continuation of U.S. application Ser.No. 13/591,566, filed on Aug. 22, 2012, which is a continuation of U.S.application Ser. No. 12/783,217, filed on May 19, 2010, which is adivisional of U.S. application Ser. No. 11/433,859, filed on May 11,2006, which claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 60/702,896 filed on Jul. 26, 2005, and entitled“Multilayered Cell Culture Apparatus” which is incorporated by referenceherein.

FIELD OF THE INVENTION

The present invention relates generally to the cellular biological fieldand, in particular, to a cell cultivating flask.

BACKGROUND OF THE INVENTION

In vitro culturing of cells provides material necessary for research inpharmacology, physiology, and toxicology. The environmental conditionscreated for cultured cells should resemble as closely as possible theconditions experienced by the cells in vivo. One example of a suitableenvironment for culturing cells is a common laboratory flask such asdemonstrated in U.S. Pat. No. 4,770,854 to Lyman. The cells attach toand grow on the bottom wall of the flask, immersed in a suitablesustaining media. The flask is kept in an incubator to maintain it atthe proper temperature and atmosphere.

Although most cells will tolerate a hydrogen ion concentration (pH)range of 6.8 to 7.8, the optimal pH for growth of mammalian cells is 7.2to 7.4. For the optimal pH to be maintained during cell cultivation, thecell culture medium must contain a buffering system. Frequently, pH ismaintained by using a bicarbonate buffering system in the medium, inconjunction with an incubator atmosphere of approximately 5 to 7 percentcarbon dioxide by volume. The carbon dioxide reacts with the water toform carbonic acid which in turn interacts with bicarbonate ions in themedium to form a buffering system which maintains the pH nearphysiological levels. Entry of carbon dioxide from the incubator intothe cell culture flask is generally achieved by using a loosely fittingor vented cap or cover so that the small opening remains for theexchange of gas between flask and incubator. Further, flasks have beensold that are made from impact resistant polystyrene plastic which ispermeable to water vapor, oxygen and carbon dioxide. However, relyingonly on the gas exchange through the polystyrene is generallyineffective since the vessel wall thickness greatly decreases thepermeability rate. Further still, flasks have been made having a cellgrowth surface that is itself an extremely thin (approximately 0.004inches thick) flexible, gas permeable membrane. While this type ofconstruction allows for gas exchange, the flexibility and thinness ofthe growth surface makes the growth of a uniform surface difficult andcontributes to problems associated with the durability of the flask.

Gas exchange, particularly the utilization of oxygen by the cells, is afactor that limits the area for cell growth within a cell culture flask.Since flasks for cell culture typically grow attachment dependent cellsin a monolayer roughly equal in size to the footprint of the flask,media volume is therefore restricted to an area within the flaskpermissive to the diffusion of oxygen. Oxygen and carbon dioxide are ofparticular importance to the culturing of cells. The supply of oxygenfor cellular respiration and metabolic function in conventional cellculture containers occupies the head space of the container, e.g., thevoid space in the container that is above the surface of the cellculture medium. Thus, the volume of the container and the surfaceswithin conventional cell culture containers are inefficiently used. Thisresults in limiting the rate of gas exchange and/or restricting theequilibration of gases. There is a need for a cell culture flask thatcan provide an increased surface area for cell growth while stillpermitting sufficient gas exchange for the multitude of attachmentdependent cells.

Desirably, many flasks are stacked together in the incubator and anumber of cultures are simultaneously grown. Small variations in thegrowth medium, temperature, and cell variability have a pronouncedeffect on the progress of the cultures. Consequently, repeatedmicroscopic visual inspections are needed to monitor the growth of thecells. As such, cell culture flasks are typically constructed ofoptically clear material that will allow such visual inspection.

With the advent of cell-based high throughput applications, fullyautomated cell culture systems have been the subject of seriousdevelopment work (see e.g. A Review of Cell Culture Automation, M. E.Kempner, R. A. Felder, JALA Volume 7, No. 2, April/May 2002, pp. 56-62.)These automated systems employ traditional cell culture vessels (i.e.common flasks, roller bottles, and cell culture dishes) and invariablyrequire articulated arms to uncap flasks and manipulate them much likethe manual operator.

There is a need for a cell culture apparatus having a rigid structurethat is capable of providing an increased surface area for cell growthwhile also providing necessary gas exchange. Even further, it isdesirable to produce a greater cell yield within commonly known flaskvolumes while permitting gas exchange at a surface of cell attachment.

Additionally, the desired cell culture apparatus will be suitable foruse in the performance of high throughput assay applications thatcommonly employ robotic manipulation.

SUMMARY OF THE INVENTION

According to an illustrative embodiment of the present invention, a cellgrowth apparatus for efficient culturing of cells is disclosed. Theillustrative apparatus includes a unitary body including a bottom traydefining a cell growth area and a top plate, connected by side walls andend walls. At least one aperture located along any periphery of theapparatus permits access to the internal volume. At least one gaspermeable substrate/membrane is affixed to a support internal to thebody of the apparatus. A tracheal space/chamber permits gases from anexternal atmosphere to be exchanged across the gas permeable, liquidimpermeable membrane, into and out of the cell culture chamber(s).Further, the tracheal space is an air chamber confined by an outervessel body. Communication between a tracheal chamber and a cell growthchamber provides a uniformity of conditions for cellular growth.Furthermore, a uniform gaseous distribution can be beneficial inproviding consistency in the culturing environment.

One embodiment of a cell growth apparatus of the present inventionincludes a plurality of cell growth chambers, each having a gaspermeable, liquid impermeable surface and an opposing surface. At leastone tracheal chamber is in communication with at least one gaspermeable, liquid impermeable surface of a cell growth chamber so thatcells can exchange gases (e.g. oxygen, carbon dioxide, etc.) with anexternal environment. The cell growth apparatus of the present inventionhas at least one tracheal chamber incorporated with a plurality of cellgrowth chambers combined into one integral unit. The integral unit thushas multiple growth surfaces in any assembled arrangement. A preferredembodiment of a cell growth apparatus of the present inventionalternates each cell growth chamber with a tracheal chamber in avertical successive orientation whereby each cell growth chamberincludes a substantially planar horizontal surface supporting the growthof attachment-dependent cells. The cell growth surface, however, may beplanar and/or nonplanar to accommodate the surface area for growth. Amodified or enhanced surface area in combination with one or moretracheal spaces enables a diversified area for growing cells.Subsequently, another embodiment of the present invention may include anarrangement of surfaces intermediary to cell growth surfaces andtracheal spaces. As such, cell growth chambers may be adjoined andconfigured so that they still have communication with a trachealchamber.

When a plurality of cell culture chambers are arranged with trachealchambers formed there-between, the tracheal chambers permit gaseousexchange between the gas permeable, liquid impermeable surface of a cellculture chamber and the external atmosphere. In a preferred embodimentof the present invention, each cell culture chamber alternates with atracheal chamber allowing the cells greater access to external gaseousexchange.

One embodiment of the apparatus of the present invention utilizes a gaspermeable, liquid impermeable membrane as the opposing surface of a cellculture chamber. In such an embodiment, a plurality of gas permeablesubstrates (internal to the body of the apparatus) can be incorporatedto increase surface area for cellular growth. Preferably then, theapparatus is capable of being rotated to facilitate the growth ofattachment-dependent cells on an alternate surface. Each gas permeablesubstrate may have a tracheal space above and/or below it. One suchembodiment is capable of incorporating one or more tracheal spacesbetween each stacked gas permeable substrate/layer. Additionally, thegas permeable membrane(s) may be treated or coated to promote cellgrowth.

Another embodiment of the present invention includes one or moresupports to form a shelf internal to the apparatus. As such, each shelfwould have at least one gas permeable substrate affixed. An alternativeembodiment may incorporate lateral ribs traversing the flask body suchthat an internal gas permeable membrane would be further capable ofsupporting cellular growth. When such supports or lateral ribs areutilized, a plurality of gas permeable membranes can be arranged orhoused within the support itself or affixed to one or more surfaces ofthe supports. It would therefore be important then, when stacking thelayers or gas permeable substrates, to include a tracheal space betweeneach layer of cell growth. Preferably, the tracheal space(s) provideuniform gaseous distribution within the cell culture chamber of theinternal apparatus. Completely filling the apparatus with media wouldallow for optimal cellular nutrient exchange. Consequently, theuniformity of conditions for cellular growth may include a determinedmedia volume per unit surface area. In another aspect, an integral unitof the cell culture apparatus comprises a plurality of modules, eachhaving a cell growth chamber and a tracheal chamber. The plurality ofmodular gas permeable substrates are utilized to permit a plurality ofcell chambers and tracheal chambers to be arranged to form one unitaryapparatus of the present invention. The plurality of layers of gaspermeable substrates are further capable of being interconnected oradjoined to provide a multiplicity of areas for cellular growth. Theplurality of modules may be interconnected in series or staggered topermit continuous flow. For easy assembly and disassembly, individualunits having snap-like features could be securely and easily adjoined.

In another embodiment of the present invention, the apparatus comprisesa manifold to access the cell growth chambers of an integral unit. Themanifold may further be capable of directing the flow of air, liquid,media and/or cellular material within the cell culture chamber.

While many embodiments of the present invention are suitable for staticcultures, another embodiment of the present invention staggers the gaspermeable substrates within the flask to permit continuous flow throughthe cell culture chamber. The staggered layers allow media tocontinuously flow or perfuse through the apparatus.

One embodiment of the present invention provides compliance withconventionally sized and shaped containers currently used such that theapparatus, device or flask of the present invention may be utilized withvarious equipment and instrumentation. Thus, the apparatus of thepresent invention may have a substantially rectangular footprint and asubstantially uniform height. The rectangular footprint would havedimensions that are substantially identical to an industry standardfootprint dimension for microplates. One embodiment of the configurationof the apparatus then may include a neck and/or cap located within thesubstantially rectangular footprint and that does not exceed the heightof the integral unit.

Another embodiment of the apparatus of the present invention maycomprise stand-offs either rising from an exterior surface of the topplate or descending from an exterior surface of the bottom tray.

For the addition and removal of media, the cell growth apparatus has atleast one access port to access multiple growth chambers. Each cellgrowth chamber, however, may have individual access ports.Supplementary, the apparatus is capable of being equipped with a septumseal accessible opening or aperture either integrated within the body ofthe apparatus itself, or as a part of a cap. When a cap is utilized, oneembodiment of the apparatus of the present invention, having a height asmeasured by the distance between an outermost plane of the bottom trayand an outermost plane of the top plate, has a cap, cover, and/or septumcovering the aperture. The cap may have a diameter that does not exceedthe height of the apparatus/flask so as to prevent interference when theflasks are stacked. Additionally, the cap may be integrally included ina top surface, side, and/or corner region of the apparatus. Theapparatus of the present invention may have an aperture which defines anentry portal and another which may define an exit portal. When gaspermeable substrates are stacked, the entry and exit portals may bepositioned in a parallel or staggered assembly so as to permit flow orperfusion through cell culture chambers within the body of theapparatus.

Convenience then dictates the utilization of one or more opticalcomponents, such as microscopic lenses, in communication with individualcell growth chambers. These lenses would allow observation of one ormore layers of cell growth. Also, and advantageously so, the apparatusis shaped and configured to enable robotic access to the interior of theapparatus without requiring cumbersome robotic arm manipulation.

The present invention also includes a method of culturing cells in theapparatus of the present invention. The method initially involvesproviding a apparatus for the growth of cells as previously described.Gas permeable substrates are first assembled into the desiredconfiguration of the apparatus followed by introduction of cells and/ormedia into the cell culture chamber of the apparatus. Thereafter, theflask can then be incubated to meet the desirable conditions for thegrowth of cells. Rotation of the apparatus further permits the culturingof cells on an alternate surface of the gas permeable substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read with the accompanying drawing figures. It is emphasized thatthe various features are not necessarily drawn to scale. In fact, thedimensions may be arbitrarily increased or decreased for clarity ofdiscussion.

FIG. 1A is a perspective external view of an illustrative embodiment ofthe apparatus of the present invention.

FIG. 1B is a cross-sectional perspective side view of an illustrativeembodiment of the present invention.

FIG. 1C is a partial internal side view of intermediary supports and gaspermeable growth surfaces of FIG. 1A.

FIG. 2 is a top view of supports utilized in another embodiment of thepresent invention.

FIG. 3 is an external top view of a frame supporting a gas permeablemembrane in another embodiment of the present invention.

FIG. 4 is a cross-sectional side view of another illustrative embodimentof the present invention.

FIG. 5 is an internal side view of the interconnected chambers of oneembodiment of the present invention.

FIG. 5A is a side view of the external frame/body of the embodiment ofFIG. 5.

FIG. 6 is an individual unit of one embodiment of the present invention.

FIG. 7 is another individual unit or tray of an embodiment of thepresent invention.

FIG. 8 is an alternative embodiment of the present invention.

FIG. 9 is another embodiment of the present invention.

DETAILED DESCRIPTION

In the following detailed description, for purposes of explanation andnot limitation, exemplary embodiments disclosing specific details areset forth in order to provide a thorough understanding of the presentinvention. However, it will be apparent to one having ordinary skill inthe art that the present invention may be practiced in other embodimentsthat depart from the specific details disclosed herein. In otherinstances, detailed descriptions of well-known devices and methods maybe omitted so as not to obscure the description of the presentinvention.

An external view of a apparatus in accordance with one embodiment of thepresent invention is shown in FIG. 1. The apparatus 100 of thisembodiment takes the form of a flask 100; the flask 100 comprises anouter vessel body 101 (see FIG. 1A) defined by a top plate 110, a bottomtray 120, sidewalls 112, and end walls 114. Disposed within the flask100 are individual cell growth chambers 111 as can be seen more clearlyin a cross-sectional illustration in FIGS. 1B and 1C. The individualcell growth chambers 111 are each defined by a generally transparentbottom surface 113 and a generally transparent top surface 115. Thesurfaces 113 and 115 are attached to the flask body 101 along thesidewalls 112 and end walls 114. Preferably, at least one bottom surface113 within each chamber 111 is gas permeable, liquid impermeablematerial and capable for the growth of cells 117. Each top surface 115is preferably a rigid, generally gas impermeable material (preferablytransparent) that will provide support to the cell growth chamber 111.In this embodiment, supports 119 allow a gas permeable membrane 113 tobe securely adhered thereto in a leak-proof sealing to the flask body101. Tracheal spaces 118 are created between each cell growth chamber111. The opposing top surface 115 of the chamber 111 defines an upperwall to the cell growth chamber 111 as well as a bottom portion of atracheal chamber 118. The tracheal chamber 118 is therefore inclusive ofa gas permeable, liquid impermeable surface 113 of a first cell growthchamber and an opposing surface 115 to a second growth chamber 111.Supports 119 further provide structural arrangements to integrallyincorporate the surfaces 113 and 115 in forming growth chambers 111 inalternation with tracheal air spaces 118 within the unitary flask 101.Each cell growth chamber 111 therefore alternates with a trachealchamber 118 in vertical successive orientation. Accessibility to thecellular growth chambers 111 is achieved via an aperture 120 within theflask body 101. The aperture 120 having a necked opening 121 isconnected to the cell growth chambers 111 via a manifold 104. Themanifold 104 is a portal for manipulation of flask contents. In thisembodiment, the necked opening 121 is covered by a cap 122 allowing theflask to be completely filled with media 127 without leakage.

In one embodiment of the present invention, the chambers 111 permitcellular growth on gas permeable membranes 113 such that multiple cellgrowth chambers 111 are integral with the body 101 of the apparatus 100and are capable of being completely filled with nutrient media for thegrowth of cells. The series of tracheal air spaces 118 through theapparatus 100 provide gaseous communication between the cells 117 of theinternal volume of the apparatus and the external environment. Thetracheal spaces 118 allow oxygenation of media located within cellgrowth chambers 111 through the gas permeable surfaces 113. Further, thetracheal chambers 118 may take the form of any air gap or space, and donot allow entrance of liquid. As a result, a rigid cell cultureapparatus 100 having multiple growth chambers 111, alternating withtracheal spaces 118, is cooperatively constructed to afford the benefitof equivalent gaseous distribution to a large volume of cells 117.Supplementary, the aperture 120 of the flask is resealable by way of aseptum and/or cap 122 to prevent contents of the flask from spilling.

The apparatus 100 of the present invention may be made by any number ofacceptable manufacturing methods well known to those of skill in theart. In a preferred method, the apparatus 100 is assembled from acollection of separately injection molded parts. Though any polymersuitable for molding and commonly utilized in the manufacture oflaboratory ware may be used, polystyrene is preferred. Although notrequired, for optical clarity, it is advantageous to maintain athickness of no greater than 2 mm.

The bottom tray 120 and top plate 110 are preferably injection molded.Various sizes and shapes of the supports 119 may be incorporated tofacilitate positioning of the membranous layers 113 for cell culture 117within the internal flask body 101. A top view of another embodiment ofthe present invention (FIG. 2) has supports 219 as elevated stand-offs219 along a frame or edge 202 of the flask 100. The supports 219 arerigid structures to support a sheet of gas permeable membrane 213adhered to the frame 202, as well as provide a structural framework toallow multiple layers (rigid or membranous 213) to be formed within theflask 200. Alternatively, FIG. 3 illustrates an inner surface 313,whereby only a portion of each cell growth chamber 300 is gas permeable.For instance, a rigid frame 302 may support a permeable membrane 313.

Gas permeable, liquid impermeable substrates 113 may be comprised of oneor more membranes known in the art. Membranes typically comprisesuitable materials that may include for example: polystyrene,polyethylene, polycarbonate, polyolefin, ethylene vinyl acetate,polypropylene, polysulfone, polytetrafluoroethylene (PTFE) or compatiblefluoropolymer, a silicone rubber or copolymer,poly(styrene-butadiene-styrene) or combinations of these materials. Asmanufacturing and compatibility for the growth of cells permits, variouspolymeric materials may be utilized. For its known competency, then,polystyrene may be a preferred material for the membrane (of about 0.003inches in thickness, though various thicknesses are also permissive ofcell growth). As such, the membrane may be of any thickness, preferablybetween about 25 and 250 microns, but ideally between approximately 25and 125 microns. The membrane 113 allows for the free exchange of gasesbetween the interior of the flask and the external environment and maytake any size or shape, so long as the membrane is supportive ofcellular growth. A preferred embodiment would include a membrane 113that is additionally durable for manufacture, handling, and manipulationof the apparatus.

The gas permeable membrane 113 is properly affixed to the supports 119by any number of methods including but not limited to adhesive orsolvent bonding, heat sealing or welding, compression, ultrasonicwelding, laser welding and/or any other method commonly used forgenerating seals between parts. Laser welding around the circumferenceof the membrane 130 is preferred to establish a hermetic seal around themembrane region such that the membrane is flush with and fused to theface of the supports 132 such it becomes an integral portion of theinterior surface of the apparatus. Once the gas permeable membrane 130is adhered, then the top plate 110 and bottom tray 120 may be joined.The parts are held together and are adhesive bonded along the seam,ultrasonically welded, or laser welded. Preferably, laser weldingequipment is utilized in a partially or fully automated assembly system.The top plate and tray are properly aligned while a laser weld is madealong the outer periphery of the joint.

Advantageously and in order to enhance cell attachment and growth, thesurfaces internal to the apparatus 100 are treated to enable cellgrowth. Treatment may be accomplished by any number of methods known inthe art which include plasma discharge, corona discharge, gas plasmadischarge, ion bombardment, ionizing radiation, and high intensity UVlight.

Finally, when a cap 122 is provided, it may be a screw cap, snap-fitcap, cap with septum, cap with air holes, or any cap known in the art.Preferably, a cap 122 is utilized in which a septum is integral with thecap 122. This will allow a cannula, tip or needle to access the contentsof the apparatus 100 without the need for unscrewing. The septum is leakproof, puncturable and capable of resealing once the needle, tip orcannula is removed from the apparatus, even after multiple punctures. Inone embodiment, the cap 122 is positioned to access the contents of theapparatus 100 via an end wall 114. As well, the cap 122 may bepositioned on a top surface 110. Additionally, the cap arrangement canalso be located such that the cap 122 does not protrude from therectangular footprint as determined by the periphery of the apparatus100. Other accessibility options may include a neck and cap arrangementwithin a corner region of the apparatus 100, such that the cap 122 wouldnot protrude from the periphery of the apparatus body 101.

In use, the apparatus 100 of the current invention is employed accordingto accepted cell growth methods. Cells are introduced to the apparatus100 though the aperture via the neck (or through a septum in theaperture). Along with the cells 117, media 127 is introduced such thatthe cells are immersed in the media. The apparatus is arranged such thatthe cell containing media covers the cell growth surfaces 113.Advantageously, the apparatus 100 is capable of being completely filledwith media since the gas permeable membranes 113 in combination with thetracheal spaces 118 provide uniform gas distribution to the cell growthsurfaces 113. This will further ensure the flow and exchange of gasesbetween flask interior and the external environment. The apparatus isthen placed within an incubator and may be stacked together with similarvessels such that a number of cell cultures are simultaneously grown.The apparatus is situated such that the bottom tray 120 assumes ahorizontal position (or vertical position depending on the cell cultureapplication). Another advantage of the apparatus 101 of the presentinvention is its enhanced capacity to grow cells on an opposing surface115 when the apparatus is rotated 180°. Thus, when the apparatus isrotated, cells can be cultured on an alternate surface 115. As such, itwould be beneficial to have the surface 115 composed of a gas permeablematerial. Where only gas permeable membranes are layered intermediary tothe apparatus, cell growth is therefore enabled on both of its gaspermeable surfaces 113/115.

Cell growth is monitored from time to time by microscopic inspectionthrough the generally transparent interior and exterior surfaces of theapparatus 100. Easier accessibility and greater visibility of cellulargrowth can be visualized when optical lenses having varyingmagnifications are employed in the external body 101. Additionally,optical lenses may be integrated within other internal surfaces of theapparatus 100.

Additionally, during the cell growth process, it may become necessary toextract the exhausted media and insert fresh media. As previouslydescribed, media replacement may be achieved through insertion of acanula, for example, through the septum. Alternatively, the media may bereplaced by removing the cap 122, in embodiments that offer this option.Once the cells are ready for harvesting, a chemical additive such astrypsin is added to the apparatus through the septum. The trypsin hasthe effect of releasing the cells from the surfaces of the apparatus.The cells can then be harvested from the flask.

A cap and neck arrangement is not necessary, however, for an apparatus400 of the present invention (FIG. 4). As illustrated in thisembodiment, supports 432 separate a series of tracheal spaces 440between each growth layer 450. The tracheal air spaces provide uniformgas distribution within the flask 400 to each cell culture layer 450. Inthis embodiment, the media in the individual cell growth chambers doesnot mix as these chambers 450 can be considered separate, and possibly,modular units 450 for easy assembly of the apparatus 400. The chambers450, however, may be interconnected via hollow supports 432. In oneembodiment, access to the interior of the apparatus 400 may beaccomplished directly, through plugged ports or apertures 460 that areon an end wall 414 to allow accessibility to each cell culture/medialayer 450. Another easy means of access may employ septa as coveringsfor the apertures 460.

Septa are capable of being integrally affixed to the body of theapparatus 400 by any of the aforementioned methods for affixing amembrane to the wall of the apparatus. The septa may take any form wellknown to those of skill in the art including a slit arrangement usefulfor blunt needles and as generally described in WO 02/066595, thecontents of which are incorporated herein by reference. Possiblematerials that may be employed in making the septa include natural andsynthetic elastomeric materials including, but not limited to siliconerubber, fluoro-carbon rubber, butyl rubber, polychloroprene rubber, asilicone elastomer composite material, thermoplastic elastomer, medicalgrades of silicone rubber, polyisoprene, a synthetic isoprene, silicone,santoprene and fluoropolymer laminate and combinations thereof. In apreferred embodiment, the elastomeric material is substantially nontoxicto cultured cells. Moreover, a universal septum may cover each aperture460 while still allowing access to each individual layer of cell growth450. This embodiment of the flask 400 may be preferred when stacking ofthe apparatus 400 is required, or when significant robotic manipulationis encountered since it eliminates the need for cap displacement.

FIGS. 5 and 5A illustrate another embodiment of the present invention.As illustrated in partial internal and external cross-sectional views,respectively, a multilayered culture vessel 501 of the present inventionis a perfusion system 500. Multiple gas permeable substrates 530 areadhered to supports/frames 532 and stacked in a parallel configurationpermitting an airway or tracheal space 540 to separate each cellulargrowth layer 550. As in previous embodiments, the gas permeablesubstrates/membranes 530 in combination with the tracheal chambers 540define the cell culture system 500. The tracheal spaces 540, alternatingwith layers of transparent gas permeable membrane(s) 530 and supports532, provide air/gas exchange with media and cell cultures 550 on analternate or opposing surface of the gas permeable substrate 530. Assuch, liquid media inside the apparatus 501 is capable of beingcontained within a layer 550. In addition, the tracheal air chambers 540under each cell growth surface 550 have gaseous communication betweenthe cells/media layers 550 and external environment via the series ofopenings 541 formed between the supports 532 in the external apparatusbody 501. The necked opening 560 comprises one aperture which defines anentry portal 562 and one aperture which defines an exit portal 564. Theentry portal 566 and exit portal 568 in conjunction with the neckedopening 560 allows access to the internal volume/layers 550 of theapparatus 500. Furthermore, in this embodiment of the apparatus/vessel501, a raised rim 580 serving as a standoff 580 is located on thesurfaces of both the top plate 510 and bottom plate 520. The standoffrim 580 is intended to contact the bottom tray 520 of an identicalvessel that is stacked on top the apparatus 501. Stacking makesefficient use of incubator space. Another attribute of having a standoffrim 580 is the allowance of an air gap between stacked flasks; the airgap is important for allowing gas exchange through any vent that may beincorporated into an upper or underside surface of the apparatus 501,and further prevents damage to the gas permeable membrane 530. Otheralternatives for standoffs 580 include raised corners, posts, ledges, orany other feature that will allow spacing between successively stackedflasks. Preferably, the bottom plate 520 is molded with a rim 580 aroundthe periphery that can engage with a standoff rim 580 from animmediately adjacent apparatus to ensure lateral stability of thestacked vessels.

For exemplary purposes and not limitation, cell seedlings, mediaexchange, and/or cell harvesting can be accessed via the entry portal(s)566 and exit portal(s) 568. In combination with the portals 566/568,linear fluid flow restrictors 564 can act as manifolds to evenly directflow during cell harvesting. Additionally, for exemplary purposes onlyand not limitation, an embodiment of the present invention incorporatesa staggered configuration of gas permeable substrates 530 in conjunctionwith the supports 532 so as to allow continuous flow or perfusionthrough the vessel 501. Various arrangements of the layers 550 andstacked substrates 530, however, would permit utilization of the vessel501 for static cell culture or cell culture in a perfusion system asdiscussed, including parallel, symmetrical, or asymmetricalarrangements.

For easier accessibility and manufacturing of the multilayered apparatus501, the arrangement of cell growth layers 550 and stacked substrates530 into individual modular units may be preferred. As such, a modularunit of one embodiment of the present invention is illustrated in FIG.6. An individual modular unit 600 comprises a support network 632 incombination with gas permeable membranes/substrates 630. A plurality ofmodular units 600 are capable of being interconnected and/or interlockedor adhered together to provide a multiplicity of growth surfaces 630/631that can be easily assembled or disassembled into a unitary multilayeredvessel for cellular culture. Vertical stacking of the modular units 600would be analogous to interconnecting building blocks. Any number ofcell growth layers 600 could be assembled or disassembled to provide awide range of accessibility options to each modular cell growth unit600. One embodiment of the present invention utilizes supports 632forming a shelf/frame 633 along a periphery of the individual unit 600in addition to lateral ribs 635 spanning or bisecting the distanceinternal to the frame 633. The transparent gas permeable substrate(s)630 are adhered to supports 632 such that air gaps or tracheal spaces640 are formed between each cell layer of gas permeable substrate 630 toallow gas distribution throughout the unitary apparatus when multipletrays are assembled into one vessel body. The tracheal spaces 640 havegaseous exchange with the external atmosphere via the trachealopenings/ports 641 in the external frame 633. Further, the trachealspaces 640 provide air/gas exchange with media and cell cultures on a[primary] surface 630 and an alternate or secondary surface 631, bothsurfaces 630/631 capable of cell growth. As seen in this embodiment,peripheral ridges or elevations 637 of the support system 632 areutilized to facilitate stacking of the modular units 600. The gaspermeable membrane 630, however, may be adhered to any of the surfacesof the support system 632 or peripheral edge 637 so as to provide aleak-proof gas permeable substrate 630 in combination with the modularunit 600 and further permitting multiple areas for cell growth on thegas permeable surfaces 630/631. Additionally, an open end 633 of theframe 632 is a feature to permit fluid flow when multiple modular units600 are stacked and adhered together into a unitary body so as to beutilized in perfusion devices. Furthermore, one embodiment of theapparatus of the present invention encompasses one gas permeablesubstrate 630 providing a primary growth surface 630, as well as an[optional] gas permeable substrate 631 providing a secondary growthsurface 631 adhered to an underside of the frame network 632.

As seen in FIG. 7, another embodiment of the present invention utilizesa modular unit 700 inclusive of a cap 762 covering an aperture 760. Amanifold 764 permits access to the internal cell culture layer 750. Aunitary cell culture chamber is capable of being constructed whenindividual units 600 and/or 700 are stacked. Further, when combined, theinternal cell culture layers 650 and/or 750 would be accessible via theaperture 760 to the unitary cell culture chamber.

In utilizing the vessels of the current invention, various methods inthe industry may be employed in accordance with accepted cell growthculturing. As discussed in a previous embodiment, cells are introducedto the flask though the neck or through the septum. Along with thecells, media is introduced such that the cells are immersed in themedia. The apparatus is arranged such that the cell-containing mediacovers the cell growth surfaces. Advantageously, the apparatus iscapable of being completely filled with media since the gas permeablemembranes in combination with the tracheal spaces provide uniform gasdistribution to the cell growth surfaces. This will furthermore ensurethe flow and exchange of gases between flask interior and the externalenvironment. The apparatus is then placed within an incubator and may bestacked together with similar flasks such that a number of cell culturesare simultaneously grown. The flask is situated such that the bottomtray assumes a horizontal position (or vertical position depending onthe cell culture application). The flask can then be rotated to permitthe culturing of cells on an alternate surface. Where only gas permeablemembranes are layered intermediary to the apparatus, cell growth isenabled on upper and under sides of the membrane (opposing gas permeablesurfaces).

Cell growth can be monitored from time to time by microscopic inspectionthrough the generally transparent surfaces. If more detailed visualinspection of the cell growth layers is required, optical lenses can beintegrated into the body or frame of the apparatus. As such, varyingmagnifications of the optical lenses would permit viewing withinindividual layers without disassembly of the apparatus. Optical lensesmay be incorporated into any surface or modular unit, as well,preferably when the units are capable of being disassembled forobservational analysis.

During the cell growth process, it may become necessary to extract theexhausted media and insert fresh media. As previously described, mediareplacement may be achieved through insertion of a canula, for example,through the septum. Alternatively, the media may be replaced by removingthe cap, in embodiments that offer this option. Once the cells are readyfor harvesting, a chemical additive such as trypsin is added to theflask through the septum. The trypsin has the effect of releasing thecells from the vessel surfaces. The cells are then harvested from theapparatus.

As discussed, the embodiments of the present invention are for exemplarypurposes only and not limitation. Supplementary, tracheal spaces arecapable of being formed above and/or below the support network when thetrays are stacked upon one another where peripheral ridges of individualmodular units permit gaps of air to flow through gas permeablesubstrates to cell growth areas when the units are interconnected. Thetracheal spaces formed within the individual units are further capableof including a diversified network of supports, intersecting and/oralternating gas permeable membrane with supports and air/trachealspaces.

The gas permeable substrates utilized in the embodiments of the presentinvention are capable of cell growth and gas exchange with the externalenvironment, achieving uniform gaseous distribution throughout the cellculture vessel. Furthermore, the apparatus of the present invention mayutilize horizontal or vertical designs having surfaces arranged foruniform gaseous distribution to cell growth areas. As seen in oneembodiment of the present invention in FIG. 8, vertical growth surfacesor gas permeable substrates 830 are separated by tracheal spaces 840.The tracheal spaces 840 allow for the exchange of oxygen, carbondioxide, and other various gases between the respiratory/gas permeablesurfaces 830 that the cells grow on and the incubator or externalatmosphere where the apparatus 800 is stored while the cells are giventime to grow. The apparatus 800 of the present invention may include acap 862 and/or a manifold 864, as well, which is unitary with the vesselbody 800.

Another embodiment of the present invention (FIG. 9) is an apparatus 900that includes an external manifold 965 allowing access to individualcell growth layers 930 via a septum as discussed previously. The units930 are modular and joined together to handle as one. Furthermore,tracheal spaces 940 allow uniform gaseous distribution to cell growthareas 930 throughout the flask 900. The uniformity of conditions forcellular growth may include a determined media volume per unit surfacearea. Though the determined ratio of volume per unit surface area haspreviously been known within a confined range of about 0.5-1.0 ml/cm²,the ratio is no longer limiting due to the direct access of the cells togaseous exchange via the gas permeable membrane upon which the cellsgrow. While efficient use of media is still preferable, any volume ofmedia may be utilized in an apparatus of this invention, the apparatusof which may be any size and/or take any shape. Further, the enhancedcapabilities of the present invention may incorporate tracheal spaces incombination with cell growth chambers into standardized orconventionally-sized containers. One embodiment of the apparatus of thepresent invention includes an increased surface area for cellular growthpreferably with a ratio of media volume per unit surface area in therange of about 0.25-0.50 ml/cm²; however, the dimensions and confinesfor cellular growth are unlimited. One such embodiment would include aheight of about 2.8 mm. As stated previously, however, the height isunrestricted so long as it permits area for the growth of cells.Furthermore, by completely filling the cell growth chambers with media,the cells have access to optimal nutrient exchange.

The embodiments of the present invention may be modified to take theshape of any device, container, apparatus, vessel, or flask currentlyused in industry. Specifically, cylindrical or alternative vessels mayutilize gas permeable substrates (internal to the vessel) in combinationwith tracheal chambers or spaces to provide an improved culturingenvironment for the growth of cells. A spiral or alternative approachinclusive of a tracheal chamber would therefore be possible. Further,although tracheal chambers may take many forms and be of any size, thepassageway-like chambers are: a) confined air spaces, b) incommunication with a gas permeable membrane that is permissive to cellgrowth, and c) communicative with the external environment via opendirect access and/or additional gas permeable membranes.

As presented, the multiple embodiments of the present invention offerseveral improvements over standard vessels currently used in industry.The improved cell culture devices remarkably enhance the volume of cellsthat are capable of being cultured in the volume enclosed by traditionalcell culture vessels. The various benefits are attributable to themulti-layered arrangement of gas permeable membranes assembled into aunitary vessel. Successive layering of individual growth chambers andtracheal chambers inclusive of the gas permeable membranes makes oxygenand other gases from the external environment available to the internalcontents of the apparatus. Specifically, gaseous exchange with thenutrient media is conducive to an even distribution of cell growth whengas permeable membranes are utilized on at least one potential growthsurface. The cell growth apparatus is capable of fully utilizing itscapacity by allowing cells access to optimal volumes of nutrient mediaand direct oxygenation via the tracheal spaces. Additional benefits areafforded to the cell culturing apparatus in which the exterior frameworkis rigidly constructed, conveniently offering easy handling, storage,and accessibility.

In one embodiment, the present invention has a footprint conforming toindustry standard for microplates (5.030+/−0.010 inches by 3.365+/−0.010inches). For this reason, the neck portion is preferably recessed withinthe overall rectangular footprint. The advantage of providing anapparatus with such a footprint is that automated equipment designedspecifically for the manipulation of microplates may be utilized withthis apparatus with very little customized modification. Similarly theheight, or the distance between the outer most portion of the bottomtray and the outer portion of the top plate, is approximately0.685+/−0.010 inches. At any rate, the present invention is not intendedto be limited in any way by the aforementioned preferred dimensions andin fact may be constructed to any dimension.

As exemplified, the apparatus may include any unitary structure, vessel,device or flask with the capacity to integrally incorporate substratesin successive orientation. The invention being thus described, it wouldbe obvious that the same may be varied in many ways by one of ordinaryskill in the art having had the benefit of the present disclosure. Suchvariations are not regarded as a departure from the spirit and scope ofthe invention, and such modifications as would be obvious to one skilledin the art are intended to be included within the scope of the followingclaims and their legal equivalents.

The invention claimed is:
 1. A cell culture vessel comprising: a topplate; a bottom tray opposed to the top plate; a manifold; and aplurality of vertically spaced and aligned cell growth chambers that arebetween the top plate and the bottom tray, each cell growth chamberdefined by a gas permeable, liquid impermeable membrane bottom surface,and a top surface, said top and bottom surfaces attached to a pair ofside walls and end walls, wherein the top surface of a first cell growthchamber and the gas permeable, liquid impermeable membrane bottomsurface of a second cell growth chamber adjacent the first cell growthchamber define a tracheal space, said tracheal space being in gaseouscommunication with the external environment through a plurality ofopenings, each opening defined by supports extending vertically betweenthe side walls and end walls of adjacent cell growth chambers, andwherein one of the end walls comprises an opening that allows fluidcommunication between the cell growth chambers and the manifold.
 2. Thecell culture vessel of claim 1, wherein the manifold comprises anaperture with a necked opening.
 3. The cell culture vessel of claim 2,wherein the aperture is covered by a cap.
 4. The cell culture vessel ofclaim 1 further comprising: one or more internal supports extending fromthe top surface of a first cell growth chamber and supporting the gaspermeable, liquid impermeable membrane bottom surface of a second cellgrowth chamber adjacent the first cell growth chamber.
 5. The cellculture vessel of claim 1, wherein each of the cell growth chambers areless than 3 mm in height.
 6. The cell culture vessel of claim 1, whereinthe gas permeable, liquid impermeable membrane bottom surface comprisesa material selected from the group consisting of polystyrene,polyethylene, polycarbonate, polyolefin, ethylene vinyl acetate,polypropylene, polysulfone, polytetrafluoroethylene (PTFE), a siliconerubber or copolymer, poly(styrene-butadiene-styrene), and anycombination thereof.
 7. The cell culture vessel of claim 1, wherein thegas permeable, liquid impermeable membrane bottom surface has athickness of 25-125 microns.
 8. The cell culture vessel of claim 1,wherein the top surface of each cell growth chamber comprises a gaspermeable, liquid impermeable membrane.
 9. The cell culture vessel ofclaim 8, wherein the gas permeable, liquid impermeable membrane topsurface comprises a material selected from the group consisting ofpolystyrene, polyethylene, polycarbonate, polyolefin, ethylene vinylacetate, polypropylene, polysulfone, polytetrafluoroethylene (PTFE), asilicone rubber or copolymer, poly(styrene-butadiene-styrene), and anycombination thereof.
 10. The cell culture vessel of claim 8, wherein thegas permeable, liquid impermeable membrane top surface has a thicknessof 25-125 microns.